It is well established that the phorbol esters, natural tumor promoters that mimic the actions of the lipid second messenger diacylglycerol (DAG), activate protein kinase C (PKC), a family of serine-threonine kinases that play important roles in multistage carcinogenesis. The traditional view of PKC as the sole receptor for the phorbol esters has been challenged by the discovery of proteins unrelated to PKC which have a Cl domain, the phorbol ester binding site in PKC. Indeed, we have established that "chimaerins", a family of proteins with homology to Rac GAPs (GTPase Activating Proteins) are high affinity receptors for phorbol esters and DAG. Moreover, like PKCs, the p2-chimaerin isoform is subject to translocation by phorbol esters in a PKC-independent fashion. Importantly, we found that 132-chimaerin accelerates GTP hydrolysis from Racl, leading to the inactivation of this GTPase. The central hypothesis of this competing renewal is that chimaerin activation by phorbol ester/DAG will impair Rac-mediated signaling and functions. In Specific Aim 1 we will explore the complex interrelationship between DAG generation by growth factors, chimaerin translocation and Rac-GTP levels. We predict that translocation of chimaerins to the plasma membrane through its C1 domain will result in the activation of chimaerin Rac-GAP activity and Rac inactivation. In Specific Aim 2 we will investigate how protein-protein interactions regulate chimaerin function, following analogous models to those described for PKC isozymes. We have isolated several chimaerin- interacting proteins that play important roles in controlling intracellular localization and function of chimaerins. A second aspect of this aim will involve the characterization of phosphotyrosine proteins isolated in our laboratory that may associate to the 132-chimaerin SH2 domain. In Specific Aim 3 we will focus on the roles of chimaerin isoforms in mitogenic signaling. Since Rac has a central role in the control of mitogenicity, our hypothesis is that chimaerins, by inhibiting Rac function, will impair mitogenic signaling and proliferation. Since Rac is critical for actin cytoskeletal reorganization, Specific Aim 4 will explore if chimaerins regulate key steps of the metastatic cascade, including filament actin membrane structures, cell spreading, migration and invasion. Our research has the potential for delineating novel PKC-independent pathways for the phorbol esters and DAG and their relationship to proliferation, malignant transformation, and metastasis. More importantly, our research challenges the use of phorbol esters as selective PKC activators in cells.